reference strains plasmids bc a1 002 gfp assay vector addgene pet 28b  (Addgene inc)

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: ( A ) Schematic representation of RNA pull-down system to identify proteins that bind UNC13A RNA. First, UNC13A RNA is in vitro transcribed from UNC13A minigene construct. Second, the RNA is labeled with a T4 RNA ligase, and the labeled RNA is then captured with streptavidin magnetic beads. The UNC13A RNA-streptavidin beads complex is mixed with either WT or TARDBP KO HeLa cell nuclei extract to elute the UNC13A RBPs, which are then assessed by western blot. ( B, C ) In vitro-transcribed RNA from WT UNC13A minigene (containing reference haplotype in UNC13A ) showed binding to TDP-43, hnRNP L, hnRNP A1, and hnRNP A2B1 by western blot ( B ) and mass spectrometry ( C ). Blots in B provided in Supporting information . Data used to generate the volcano plot in C can be found in . ( D ) In vitro-transcribed RNA from WT UNC13A minigene demonstrate binding of UNC13A cryptic exon to hnRNP L, hnRNP A1, and hnRNP A2B1 even in the absence of TDP-43 ( TARDBP KO HeLa cells), as shown in western blot. Blots in D provided in Supporting information . TARDBP KO HeLa cells treated with siRNAs against HNRNPL , HNRPNA1 , and HNRNPA2B1 were used as an additional negative control in the assay. Representative images of at least 2 independent experiments are shown. RBP, RNA-binding protein; siRNA, small interfering RNA; TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: In Vitro, Construct, Labeling, Magnetic Beads, Western Blot, Binding Assay, Mass Spectrometry, Negative Control, RNA Binding Assay, Small Interfering RNA

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: In vitro-transcribed RNA from WT and CE SNP UNC13A minigenes were incubated with nuclear extracts from WT HeLa cells to assess their ability to bind the following proteins by western blot analyses after pull-down: TDP-43 ( A ), hnRNP L ( B ), hnRNP A1 ( C ), and hnRNP A2B1 ( D ). The graphs show reduced binding to CE SNP minigene by TDP-43 and other hnRNPs, as quantified by the signal intensity of the western blots using Image J. Graphs represent mean ± SEM of 6 independent assays. Statistical differences were assessed by Student’s t test, ** P < 0.005, **** P < 0.0001. Blots provided in Supporting information . Data used to generate graphs can be found in . CE, cryptic exon; SEM, standard error of mean; SNP, single-nucleotide polymorphism; TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: In Vitro, Incubation, Western Blot, Binding Assay

Journal: PLOS Biology

Article Title: TDP-43 and other hnRNPs regulate cryptic exon inclusion of a key ALS/FTD risk gene, UNC13A

doi: 10.1371/journal.pbio.3002028

Figure Lengend Snippet: ( A ) WT UNC13A minigene was expressed in WT HeLa cells treated with either control (siControl) or siRNAs against TARDBP (siTARDBP), HNRNPL (siHNRPL), HNRNPA1 (siHNRNPA1), or HNRNPA2B1 (siHNRNPA2B1). RNA was extracted, and RT-qPCR was performed to assess the expression levels of UNC13A cryptic (A), TARDBP , HNRNPL , HNRNPA1 , or HNRNPA2B1 RNA. (B, C) Flag-tagged TDP-43, hnRNP L, hnRNP A1, and hnRNP A2B1 were expressed in TARDBP KO HeLa cells transfected with UNC13A WT or CE SNP minigenes to evaluate the ability of other hnRNPs to repress UNC13A cryptic exon inclusion by RT-qPCR. A representative immunoblot confirming the expression of each Flag-tagged plasmid using a Flag antibody is shown in B. Blot provided in Supporting information . All graphs represent mean ± SEM of UNC13A cryptic RNA levels of 3 independent experiments. Statistical differences were assessed by one-way followed by Tukey’s multiple comparisons test (A) or two-way (C) ANOVA (ns: not significant, * P < 0.05, ** P < 0.005, **** P < 0.0001). Data used to generate graphs can be found in . CE, cryptic exon; hnRNP, heterogeneous nuclear ribonucleoprotein; SEM, standard error of mean; siRNA, small interfering RNA; SNP, single-nucleotide polymorphism;TDP-43, TAR DNA-binding protein-43.

Article Snippet: To generate Flag-tagged hnRNP A1 and hnRNP A2B1 overexpression constructs, an hnRNP A2B1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC000506) and an hnRNP A1 protein vector (pPM-N-D-C-HA) (Applied Biological Materials, Accession Number BC002355) were used.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Plasmid Preparation, Small Interfering RNA, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: microRNA-196a attenuates ischemic brain injury in rats by directly targeting high mobility group A1

doi: 10.3892/etm.2019.7152

Figure Lengend Snippet: Expression of miR-196a and HMGA1 in cortical neurons subjected to OGD. (A and B) TUNEL was used to evaluate the apoptosis of cortical neurons following OGD (magnification, ×100). Blue fluorescence indicates nuclei and green fluorescence indicates apoptotic cells. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of (C) miR-196a and (D) HMGA1. (E) Protein expression levels of HMGA1 were determined using western blot analysis. Data are expressed as the mean ± standard deviation (n=8). *P<0.05 and **P<0.01 vs. control. HMGA1, high mobility group A1; ODG, oxygen-glucose deprivation; miR-196a, microRNA-196a; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: A total of 2×10 5 cortical neurons were plated in 6-well plates and cultured at 37°C for 24 h. miR-196 mimics, miR-196 inhibitor or high mobility group A1 (HMGA1) vectors were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 30 mM.

Techniques: Expressing, TUNEL Assay, Fluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Control, End Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: microRNA-196a attenuates ischemic brain injury in rats by directly targeting high mobility group A1

doi: 10.3892/etm.2019.7152

Figure Lengend Snippet: miR-196a reduced neuronal apoptosis and infarction following ischemic brain injury in vivo . (A) Rats were pretreated with a miR-196a antagomir, antagomir control and subsequently subjected to 1.5 h MCAO and 24 h reperfusion. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of miR-196a in rat brain tissues. (B) Coronal sections exhibiting the different treatments were stained with 1% 2,3,5-triphenyltetrazolium chloride and were evaluated. The infarct region lacked staining and appeared white, whereas the normal non-infarct tissue appeared red. (C) The infarct volume was determined and (D and E) TUNEL assay was performed to evaluate the apoptosis of brain tissue following MCAO (magnification, ×100). Blue fluorescence indicates nuclei and green fluorescence indicates apoptotic cells. (F and G) Immunocytochemistry was used to evaluate the HMGA1 expression in brain tissue. Positive nuclei were indicated as brown in color. Data are expressed as the mean ± standard deviation (n=8). *P<0.05 and **P<0.01 vs. the sham group; # P<0.05 vs. the MCAO group. HMGA1, high mobility group A1; miR-196a, microRNA-196a; MCAO, middle cerebral artery occlusion; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling.

Article Snippet: A total of 2×10 5 cortical neurons were plated in 6-well plates and cultured at 37°C for 24 h. miR-196 mimics, miR-196 inhibitor or high mobility group A1 (HMGA1) vectors were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 30 mM.

Techniques: In Vivo, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Staining, TUNEL Assay, Fluorescence, Immunocytochemistry, Standard Deviation, End Labeling

Journal: Experimental and Therapeutic Medicine

Article Title: microRNA-196a attenuates ischemic brain injury in rats by directly targeting high mobility group A1

doi: 10.3892/etm.2019.7152

Figure Lengend Snippet: miR-196a directly targeted HMGA1 and inhibited its expression. (A and B) The 3′UTR reporter assay was performed and miR-196a was overexpressed in coronal neurons. pGL3-HMGA1-3′-UTR-WT or pGL3-HMGA1-3′-UTR-Mut was co-transfected with pRL-TK using Lipofectamine 2000. *P<0.05 as indicated. (C) Reverse transcription-quantitative polymerase chain reaction analyses of miR-196a level in different treatment groups. (D) Western blot analysis was used to access the protein expression of neuron cells exposed to different treatment. Data are expressed as the mean ± standard deviation (n=8). *P<0.05 and **P<0.01 vs. control; # P<0.05 vs. the OGD group. WT, wild-type; MUT, mutant; UTR, untranslated region; HMGA1, high mobility group A1; miR-196a, microRNA-196a; OGD, oxygen-glucose deprivation; 3′UTR, 3′untranslated region.

Article Snippet: A total of 2×10 5 cortical neurons were plated in 6-well plates and cultured at 37°C for 24 h. miR-196 mimics, miR-196 inhibitor or high mobility group A1 (HMGA1) vectors were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 30 mM.

Techniques: Expressing, Reporter Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Control, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: microRNA-196a attenuates ischemic brain injury in rats by directly targeting high mobility group A1

doi: 10.3892/etm.2019.7152

Figure Lengend Snippet: HMGA1 reversed the induction of apoptosis by miR-196a. (A and B) TUNEL assay was used to evaluate the apoptosis of cortical neurons exposed to different treatments, (magnification, ×100). Blue fluorescence indicates nuclei and green fluorescence indicates apoptotic cells. Data are expressed as the mean ± standard deviation (n=8). *P<0.05 and **P<0.01 vs. control; # P<0.05 vs. the OGD group; $ P<0.05 vs. the miR-196a group. HMGA1, high mobility group A1; miR-196a, microRNA-196a; OGD, oxygen-glucose deprivation; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling.

Article Snippet: A total of 2×10 5 cortical neurons were plated in 6-well plates and cultured at 37°C for 24 h. miR-196 mimics, miR-196 inhibitor or high mobility group A1 (HMGA1) vectors were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at a final concentration of 30 mM.

Techniques: TUNEL Assay, Fluorescence, Standard Deviation, Control, End Labeling